Hydrogen Production from Barley Straw and Miscanthus by the Hyperthermophilic Bacterium, Cadicellulosirupter bescii

This work aimed to evaluate the feasibility of biohydrogen production from Barley Straw and Miscanthus. The primary obstacle in plant biomass decomposition is the recalcitrance of the biomass itself. Plant cell walls consist of cellulose, hemicellulose, and lignin, which make the plant robust to decomposition. However, the hyperthermophilic bacterium, Caldicellulosiruptor bescii, can efficiently utilize lignocellulosic feedstocks (Barley Straw and Miscanthus) for energy production, and C. bescii can now be metabolically engineered or isolated to produce more hydrogen and other biochemicals. In the present study, two strains, C. bescii JWCB001 (wild-type) and JWCB018 (ΔpyrFA Δldh ΔcbeI), were tested for their ability to increase hydrogen production from Barley Straw and Miscanthus. The JWCB018 resulted in a redirection of carbon and electron (carried by NADH) flow from lactate production to acetate and hydrogen production. JWCB018 produced ~54% and 63% more acetate and hydrogen from Barley Straw, respectively than its wild-type counterpart, JWCB001. Also, 25% more hydrogen from Miscanthus was obtained by the JWCB018 strain with 33% more acetate relative to JWCB001. It was supported that the engineered C. bescii, such as the JWCB018, can be a parental strain to get more hydrogen and other biochemicals from various biomass.


Introduction
In the near future, we will be required to contend with serious environmental problems such as the greenhouse effect, global climate change, and fine dust caused by the widespread use of fossil fuels.Fuel production from plant biomass is a potential remedy for many of these problems.However, plants have evolved to resist decomposition by microorganisms, and plant cell walls consist of cellulose, hemicellulose, and lignin, which can make the plant recalcitrant [1][2][3][4].Caldicellulosiruptor bescii is able to utilize lignocellulosic feedstocks efficiently and is also the most thermophilic/cellulolytic bacterium, with an optimal growth temperature of 70°C ~ 80°C [5][6][7].C. bescii also can utilize C5 and C6 sugars released from plant biomass.The carbohydrates are oxidized in the Embden-Meyerhof-Parnas pathway producing acetate, lactate, CO 2 , and hydrogen as major fermentative end products (Fig. 1) [7][8][9].In this pathway, pyruvate is the major metabolic branch point during fermentation, routing carbon to lactate or acetyl-CoA and electrons carried by nicotinamide adenine dinucleotide (NADH) to lactate or H 2 .At this point, the acetate production pathway is essential for H 2 production, permitting the re-oxidation of NADH and ferredoxin, which can be simultaneously oxidized by a bifurcating hydrogenase [9].Hydrogen is currently the most actively studied biofuel of these fermentative end products.There are already many bacterial strains that produce high yields of hydrogen, especially Thermoanaerobacter tengcongensis (~ 4.0 mol/ mol glucose, [10]), Thermotoga maritima (~ 4.0 mol/mol glucose, [11]), Thermococcus kodakaransis (~ 3.3 mol/mol glucose, [12]), etc.Although C. bescii has special advantages for the conversion of plant biomass to fuels and chemicals, this strain also possesses a strong Restriction-Modification (R-M) system, which fundamentally limits DNA transformation and, in turn, limits the practicality of using this strain for commercial purposes at scale [13].To overcome the R-M system in C. bescii, Chung, et al. constructed a mutant strain, JWCB018, by deleting the endonuclease encoding gene (cbeI), rendering this strain more easily engineered [13].C. bescii is an attractive platform for metabolic engineering for maximal H 2 production from various plant biomass (especially Barley Straw and Miscanthous), partly because it could significantly reduce processing costs compared to current fuel production method from biomass.
Here, we provide evidence of highly efficient H 2 production by C. bescii by comparing a C. bescii wild-type, JWCB001, and a mutant strain, JWCB018 (ΔpyrFA ΔldhΔ cbeI).It will be feasible to utilize C. bescii to efficiently produce a significant amount of H 2 from plant biomass by reprogramming the bioenergetic pathways of C. bescii, such as altering the acetate production towards H 2 production.

Procurement and Validation of C. bescii Strains
A mutant strain (ΔpyrFA ΔldhΔ ΔcbeI), JWCB018, was constructed by Chung, et al. [13], and it spontaneously became a double mutant (ΔcbeI Δldh) by an active transposon on the lactate dehydrogenase-encoding gene (ldh) [14].The ΔcbeI deletion strain was constructed based on JWCB005, which was a pyrFA-deleted strain of C. bescii [15].Cha et al. isolated and purified the double mutant [14].The two C. bescii strains, a wild-type (JWCB001) and a mutant (ΔpyrFA ΔcbeI Δldh, JWCB018), were obtained from Dr. Janet Westpheling's Lab at the University of Georgia, USA (Table 1).The strains were stored in 10% DMSO (dimethyl sulfoxide) at -80°C immediately upon receipt.Deletions were confirmed by Polymerase Chain Reactions (PCRs) with specific sets of primers.To confirm a cbeI deletion, an external PCR and an internal PCR of cbeI were performed with primer sets, MC011/ MC012 and MC011/MC013, respectively (Table 1).To confirm ldh interruption for modification by transposon insertion, an external PCR of ldh was performed with a primer set, MC014/MC015 (Table 1).All PCR products were evaluated by gel electrophoresis and visualized on 0.8% agarose gels.

Growth Media and Conditions
C. bescii JWCB001 and JWCB018 (Table 1) were anaerobically grown in low osmolarity defined (LOD) medium [16] containing 40 μM MOPS with a final pH of 7.0, supplemented with cellobiose (1.0% (wt/vol); catalog no.01407; Chem-Impex, USA) or maltose (1.0% (wt/vol); catalog no.70090-0401; Junsel, Korea) as the carbon source, unless otherwise noted.The liquid cultures for the two strains were grown from a 2% inoculum, then  incubated at 75°C in anaerobic culture bottles degassed with 7 cycles of vacuum and argon.An auxotrophic mutant, JWCB018, was grown in LOD medium supplemented with 40 μM uracil.

Biomass Pretreatment
Hordeum vulgare (Barley straw) and Miscanthus (Miscanthus) were obtained from Jeonnam, Korea in 2021.The air-dried biomass was chopped to a length of 5 cm using a tub grinder (Tomotech Ltd., Korea).The chopped biomass was then ground using a 20 hp hammer mill (Sunbrand Industrial Inc., Korea) with 3.0 mm screens, dried at 60°C for 24 h, and then stored in desiccators.The chemical analysis indicated that the biomass mainly consisted of 35-43% cellulose, 22-25% hemicellulose, 18-22% lignin, and 3-7% ash.
Biomass was pretreated in an 800 ml pressure vessel equipped with a temperature and pressure sensor.The mixture of biomass and alkali catalyzed organic solvent (1:9, 300 ml working volume) was then loaded into the vessel.The alkali catalyzed organic solvent contained 12.2% sodium hydroxide per dried biomass volume in 57.4% ethanol.The vessel was then heated to 163°C for 60 min.Nitrogen gas was additionally loaded to 6 MPa in the vessel for explosion before the pretreated biomass was collected into a separator via pressure and temperature differences.The solid hydrolysate was obtained using a Buchner funnel with a 10 μm nylon filter and neutralized with tap water.

Determination of Cell Growth, Carbon Sources, and By-Products during Fermentation
The strains were grown in stoppered 125-ml serum bottles containing 50 ml LOD medium supplemented with different carbon sources.Medium for the JWCB018 was supplemented with 1 mM uracil.Duplicate bottles were inoculated with a fresh 2% (vol/vol) inoculum and incubated at 75°C with shaking at 150 rpm.Optical cell density was monitored using a Biomate5 UV-visible spectrophotometer (Thermo Fisher, USA) measuring absorbance at 680 nm.The C. bescii wild-type and mutant strain were incubated in the same culture conditions, supplemented with 10 g/l (wt/vol) cellobiose, 20 g/l (wt/vol) Avicel (catalog no.11365; Sigma-Aldrich, USA), 10 g/l (wt/vol) pretreated Barley Straw and 10 g/l (wt/vol) pretreated Miscanthus as a single carbon source, respectively.
Fermentative products, cellobiose, acetate, and lactate were analyzed on an Agilent Technologies 1200 Series HPLC system (Agilent Technologies, USA).Metabolites were separated on a Rezex ROA-Organic Acid H+ (8%) column (Phenomenex, USA) under isocratic temperature (60°C).Five mM H 2 SO 4 was used as a mobile phase at a flow rate of 0.6 ml/min, and then the samples were passed through a refractive index detector (Agilent 1200 Infinity Refractive Index Detector).Identification of separated chemicals was compared to retention times with standards, and total peak areas were integrated and compared with peak areas and retention times of known standards for each compound of interest.

Determination of H 2 Production
The culture bottles were cooled to room temperature after 48 h of incubation at 75°C, and H 2 was separated on an Agilent Technologies 8890 GC system (Agilent), equipped with a thermal conductivity detector (TCD) at 200°C and N 2 reference flow, using the Agilent J&W CP-Molsieve 5Å CP 7535 column (Agilent) at 30°C.The hydrogen peak was isolated by comparing retention times with H 2 standards.To measure H 2 concentration produced, total peak areas were integrated and compared to peak areas and retention times of known H 2 standards.

Comparison of Growth and Hydrogen Production in the C. bescii Strains
Growth rates of C. bescii JWCB001 and JWCB018 were compared when strains were cultured in LOD media [16] supplemented with 1.0% cellobiose (Fig. 3A).The growth rate of JWCB018 in the exponential phase was indistinguishable from that of the wild-type, JWCB001, although the final growth of the mutant strain, JWCB018, revealed a ~ 10% lower cell density than the wild-type (Fig. 3A).

Comparison of the Final Fermentation Products and Carbon Balances of C. bescii Wild-Type and Mutant Strains
To compare the final fermentation products, C. bescii wild-type and mutant strains were grown in LOD medium [16] with 1% (wt/vol) cellobiose, 2% (wt/vol) Avicel, 1% (wt/vol) Barley Straw, or 1% (wt/vol) Miscanthus as a carbon source, respectively.The fermentation products were monitored by HPLC over the course of 48 h (Fig. 4), and final carbon utilization was calculated (Table 2).Product yield was calculated as product yield per mole cellobiose (mol/mol).The HPLC analysis showed that the C. bescii wild-type, JWCB001, produced lactate (6.5 mM from cellobiose, 10.5 mM from Avicel, 12.3 mM from Barley Straw, and 8.8 mM from Miscanthus) and acetate (12.2 mM from cellobiose, 10.6 from Avicel, 14.1 mM from Barley Straw, and 11.9 mM from Miscanthus) by the end of the time course (48 h, Fig. 4).The mutant strain (JWCB018, ΔldhΔcbeI) did not produce lactate over the same time frame.However, it produced more acetate (17.7 mM from cellobiose, 18.3 mM from Avicel, 21.6 mM from Barley Straw, and 20.2mM from Miscanthus) than wild-type by the end of the time course (48 h, Fig. 4).The JWCB018 (ΔpyrFA Δldh ΔcbeI) produced more acetate than wild-type, JWCB001, because its carbon flow to acetate was increased by the inactivation of ldh (Fig. 4).
The carbon mass balance for the end products of growth on 1% cellobiose was calculated at the end of the time course (48 h, Table 2).The wild-type yielded 1.2 mol/mol lactate, 2.2 mol/mol acetate, and 1.8 mol/mol hydrogen, with 94% overall carbon recovery (Table 2).The mutant strain, JWCB018 (ΔpyrFA Δldh ΔcbeI), did not produce lactate at all and yielded 2.9 mol/mol acetate and 2.1 mol/mol hydrogen, with 90% carbon recovery overall (Table 2).

Discussion
Based on the genome sequence of C. bescii, there is only one predicted lactate dehydrogenase gene (Cbes1918).To confirm this, both JWCB001 and JWCB018 were grown on different carbon sources.The JWCB018 mutant (Cbes1918 expression interrupted by an active transposon (Figs.2B and 2D)) produced no detectable lactate.On  the other hand, the wild-type, JWCB001, demonstrated lactate productions of 6.5 mM on cellobiose, 10.6 mM on Avicel, 12.3 Mm on Barley Straw, and 8.8 mM on Miscanthus (Fig. 4).Instead of lactate, the JWCB018 mutant strain produced much more acetate and hydrogen due to increased carbon and electron (carried by NADH) flux to acetate and hydrogen, respectively.To compare the production of acetate and hydrogen, C. bescii JWCB001 and JWCB018 were grown in LOD medium [16] with soluble cellobiose or real-world biomass (Barley Straw and Miscanthus) as a carbon source.JWCB018 produced 54% more acetate and 25% more hydrogen than JWCB001 when both strains were grown on 1% cellobiose for 48 h (Figs. 3 and 4).When the strains, JWCB001 and JWCB018, were grown on 1% Barley Straw as the sole carbon source, they showed a very similar profile to that of cellobiose.The Δldh strain, JWCB018, showed 54% and 63% more acetate production and 33% and 25% hydrogen production on Barley Straw and Miscanthus, respectively, than JWCB001 (Figs. 3 and 4).The JWCB018 produced more acetate and hydrogen on 1% Barley Straw and Miscanthus than on 1% cellobiose because the plant biomass (Barley Straw and Miscanthus) consists of cellulose, hemicellulose, and lignin, which can all be effectively decomposed by C. bescii [5][6][7].
In this study, we provide evidence for the effective production of biohydrogen from the real-world biomass by C. bescii strains.Since the endonuclease-encoding gene (cbeI) in C. bescii mutant strain was deleted, no R-M system exists in the cells.This permits easy metabolic engineering of the strain to optimize its hydrogen production from the real-world biomass.Other effective modifications could include eliminating the acetate production by deletion of ak and pta genes coding for key enzymes in the acetate biosynthetic pathway, or heterogeneous expression of strong hydrogenases from other thermophiles.Alternatively, a strong promotor could be inserted to amplify H 2 production.Due to its versatility, the C. bescii mutant strain, JWCB018, lends itself well to rational strain engineering and can serve as a parent strain for production of biohydrogen at scale from lignocellulosic feedstocks.

Fig. 3 .
Fig. 3. Comparison of growth (OD 680 nm ) and hydrogen production for the wild-type, JWCB001, and the mutant, JWCB018, strains.(A) Growth of C. bescii strains on 1% cellobiose as a sole carbon source; blue square, JWCB001; black circle, JWCB018 (ΔpyrFA Δldh ΔcbeI).(B) Hydrogen production on different carbon sources by each strain at the end of incubation (48 h); blue bars, JWCB001; black bars, JWCB018.Error bars based on two biologically independent experiments.

Table 1 . Strains and primers used in this study.
MC013 CAA CGT GGT GAT GTA AGA GAT ATG TTA GC This study MC014 ATC TTG CCA CGT ACA ATC TCT CCT TCA G This study MC015 TCT CTG ATA ATA TGG CCC AGG AGA TTA TTC TTC This study 1 German collection of microorganisms and cell cultures.